Project Summary: Transcription factors of the DAF-16/FOXO family are evolutionarily conserved master regulators capable of integrating diverse environmental stimuli and coordinating development, metabolism, stress responses, and longevity. Deregulation of FOXO proteins in humans is well known to play a key role in age-related diseases such as cancer and diabetes. For DAF-16/FOXOs to achieve their multi-faceted and central roles, their transcriptional activities need to be exquisitely regulated;however, the mechanisms underlying the regulation of DAF-16/FOXO-mediated gene transcription are largely unknown. The long-term goal of this proposal is to elucidate comprehensively how DAF-16/FOXO transcriptional activities are regulated in the nucleus and how this regulation contributes to longevity determination. We recently discovered that the C. elegans ortholog of the nuclear protein host cell factor 1 (HCF-1) modulates longevity by functioning as a DAF-16 co-factor that inhibits DAF-16-mediated gene regulation. Since HCF-1 is the first DAF-16 negative co-factor reported in C. elegans, this finding provides an important entry point for further investigation of how DAF-16 transcriptional activities are regulated. In Aim 1, we propose to test the hypothesis that HCF-1 functions in a temporally and spatially regulated manner to modulate lifespan. Since HCF-1 represents a new longevity factor, it will be critical to determine when and where it functions to modulate C. elegans lifespan. In Aim 2, we propose to test the hypothesis that the interplay between HCF-1 and other DAF-16 co-factors determines DAF-16-mediated transcriptional outputs. HCF-1 is a DAF-16 co-factor that inhibits DAF-16-mediated gene regulation. It is likely that HCF-1 will regulate DAF-16 transcriptional activities by functionally interacting with some of the DAF-16 co-factors that are known to promote DAF-16 transcriptional activities, such as SIR-2.1, SMK-1, BAR-1. In Aim 3, we propose to test the hypothesis that additional nuclear regulators of DAF-16-mediated gene transcription are also important longevity factors. Thus far, four DAF-16 co-factors (HCF-1, SIR-2.1, SMK-1, BAR-1) have been reported. Considering that DAF-16 participates in diverse biological processes, it is very likely that DAF-16 transcriptional activities are regulated by many more nuclear factors than are currently known. Since DAF-16 and its co-factors (HCF-1, SIR-2.1, SMK-1, BAR-1) are all highly conserved, findings from our studies will provide important insights into FOXO regulation and longevity determination in mammals. Moreover, the mammalian orthologs of DAF-16 and its co-factors are known to play key roles in the pathogenesis of a number of human diseases, such as cancer and diabetes, findings from our studies may inspire future therapeutic development aiming to alleviate some of these age-related pathologies in humans.